In situ real-time monitoring of upconversion emission is applied to study the reaction mechanism for the synthesis of β-NaYF4:17% Yb,3% Er nanoparticles in oleic acid and octadecene via the heat-up method. Transmission electron microscopy is used to correlate the spectroscopic signature of the reaction mixture with its composition. The power of real-time spectroscopic monitoring to precisely time the duration of the various stages of the reaction, and to accurately identify the transitions between those stages, including the completion of the reaction, is demonstrated. During the heat-up stage, the initial precipitate present is transformed into small α-phase nanoparticles. In the second and longest stage of the reaction, a period of relative stasis is maintained, during which there is at most a slight change in the size distribution of the α-phase nanoparticles formed during heat-up. In the third stage, a relatively rapid conversion of small α-phase nanoparticles to the larger β-phase product nanoparticles is observed. The size distribution of the β product remains relatively constant during the phase-transition stage, indicating that, once the β particles begin to form, they grow very rapidly to the final product size. A significant variation in the time required to complete the reaction is observed, which is due almost entirely to the variation in the duration of the second reaction stage. The time required to complete the reaction does not appear to affect the size or uniformity of the product β-NaYF4:17% Yb,3% Er nanoparticles.

BackgroundErythropoietin (EPO), a pleiotropic cytokine, binds to its receptor (EPOR) in bone marrow, activating a signaling cascade that results in red blood cell proliferation. A recently discovered naturally occurring EPO mutation (R150Q) at active site 1 (AS1) of the protein was shown to attenuate its canonical downstream signaling, eliminating its hematopoietic effects and causing a fatal anemia. The purpose of this work was to analyze the EPO–EPOR complex computationally to provide a structural explanation for this signaling change.Materials and methodsComputational structural biology analyses and molecular dynamics simulations were used to determine key interaction differences between the R150Q mutant and the wild-type form of EPO. Both were compared to another variant mutated at the same position, R150E, which also lacks hematopoietic activity.ResultsThe ligand–receptor interactions of the R150Q and R150E mutants showed significant variations in how they interacted with EPOR at AS1 of the EPO–EPOR complex. Both lost specific reported salt bridges previously associated with full complex activation.ConclusionThis work describes how the ligand–receptor interactions at AS1 of the EPO– EPOR complex respond to mutations at the 150th position. The interactions at AS1 were used to propose a potential mechanism by which the binding of EPO to the extracellular domain of EPOR influences its cytosolic domain and the resulting signaling cascade.

Introduction: Carbamoylated erythropoietin (CEPO) is a chemically engineered, nonhematopoietic derivative of erythropoietin (EPO) that retains its antidepressant and procognitive effects, which are attributed to the increased expression of neurotrophic factors like brain derived neurotrophic factor (BDNF), in the central nervous system. However, the chemical modification process which produces CEPO from erythropoietin (EPO) requires pure EPO as raw material, is challenging to scale-up and can also cause batch-to-batch variability. To address these key limitations while retaining its behavioral effects, we designed, expressed and analyzed a triple, glutamine, substitution recombinant mimetic of CEPO, named QPO. Methods and Materials: We employ a combination of computational structural biology, molecular, cellular and behavioral assays to design, produce, purify and test QPO. Results: QPO was shown to be a nonhematopoietic polypeptide with significant antidepressant-like and pro-cognitive behavioral effects in rodent assays while significantly upregulating BDNF expression in-vitro and in-vivo. The in-silico binding affinity analysis of QPO bound to the EPOR/EPOR homodimer receptor shows significantly decreased binding to Active Site 2, but not Active Site 1, of EPOR. Discussion: The results of the behavioral and gene expression analysis imply that QPO is a successful CEPO mimetic protein and potentially acts via a similar neurotrophic mechanism, making it a drug development target for psychiatric disorders. The decreased binding to Active Site 2 could imply that this active site is not involved in neuroactive signaling and could allow the development of a functional innate repair receptor (IRR) model. Substituting the three glutamine substitution residues with arginine (RPO) resulted in the loss of behavioral activity, indicating the importance of glutamine residues at those positions.